During the past year we have continued to employ both biochemical and genetic approaches to the study of the acquisition of glucocorticoid resistance in human leukemic cells. We have identified a new steroid-resistant phenotype in CEM-C7 cells designated activation-labile molybdate-resistant. Glucocorticoid receptors displaying this phenotype are unable to assume or maintain the activated form of the steroid-receptor complex. In contrast to previous activation labile mutants we have isolated, the steroid receptors of this phenotype cannot be stabilized by sodium molybdate during attempted activation. Somatic cell hybrids constructed between cells of this phenotype and wild-type cells or other steroid resistant cells demonstrate that all steroid resistant phenotypes belong to the same genetic complementation group. Thus, all steroid-resistant phenotypes observed in CEM-C7 derived clones appear to be the result of alterations within the receptor locus itself. We have developed a high resolution two-dimensional gel electrophoresis system for the analysis of affinity-labeled glucocorticoid receptors. Using this system we have determined that there is charge heterogeneity in the 90 kilodalton steroid-binding component of normal glucocorticoid receptors. Two distinct species of apparent pI 7.0 and 6.4 have been identified for the unactivated form of the affinity-labeled human glucocorticoid receptor. We are currently investigating the origins of this heterogeneity as well as applying this technique to the physical analysis of mutant receptors. (D)